Pseudomonas aeruginosa infections result in significant morbidity and mortality in people suffering from cystic fibrosis (CF). A key feature of these infections is that they involve biofilm communities. Another is that during the course of chronic infection, P. aeruginosa acquires mutations that help it adapt to the CF environment. This proposal will investigate a clear link between biofilm formation and genetic adaptation, the RSCV phenotype. RSCVs generally exhibit increased biofilm formation and antimicrobial tolerance relative to the non- mucoid, smooth colony morphotype. One important trait of RSCVs are elevated production of the biofilm matrix exopolysaccharide PEL. The structure of PEL remains unknown, and its elucidation is a goal of this proposal. In addition, P. aeruginosa RSCVs are actively selected for during the course of chronic cystic fibrosis (CF) airway infections. We recently discovered that the prevalence of RSCVs in the CF airways has been grossly underestimated and we subsequently identified novel genetic loci linked to the RSCV phenotype. The molecular mechanism underpinning why these mutations produce RSCVs is unclear. Finally, we found that RSCVs can be highly variable phenotypically, depending upon the causal mutation. Some of these variable phenotypes are pathogenically relevant, suggesting that different RSCVs may vary in their ability to form biofilms and cause chronic infection. The overall goals of this application are to determine the structure of PEL and to elucidate the molecular mechanism leading to the RSCV phenotype that results from novel RSCV- linked mutations. We will also examine the pathogenic fitness advantages of different RSCV genotypes using chronic animal models of infection and in vitro cellular immunological assays. Once mucoid genetic variants of P. aeruginosa emerge, prognosis of CF patients clearly worsens. Thus, there exists a critical window of opportunity for immunological or chemotherapeutic intervention prior to mucoid conversion. RSCVs are usually observed prior to mucoidy, thus therapeutic strategies taking advantage of this window of opportunity would have to account for them. A thorough analysis of RSCVs and Pel may lead to the development of therapeutic agents and improve the quality of life for CF patients as well as individuals with other P. aeruginosa infections that involve biofilms.